The cell plasma membrane represents an efficient barrier that prevents most molecules that are not actively imported from cellular uptake, thus also hampering the targeted delivery of therapeutic substances. Only a small range of molecules having a particular molecular weight, polarity and/or net charge is able to (passively) diffuse through cell membranes. All other molecules have to be actively transported, e.g., by receptor-mediated endocytosis or via ATP-binding transporter molecules. In addition, molecules may also artificially be forced to pass the cell membrane, for example by means of electroporation, cationic lipids/liposomes, micro-injection, viral delivery or encapsulation in polymers. However, these methods are mainly utilized to deliver hydrophobic molecules. Furthermore, the significant side effects associated with these methods and the fact that their applicability is mostly limited to in vitro uses has prevented them from becoming an efficient tool for the delivery of drugs or other therapeutically active agents to cells in order to prevent or treat medical conditions.
In particular, the requirement of targeted delivery has also turned out to represent a major challenge in the development of RNAi (RNA interference)-based drugs. Such agents comprise small RNA molecules (e.g., siRNAs, miRNAs or shRNAs) that interfere with the expression of disease-causing or disease-promoting genes. Following the demonstration of RNAi in mammalian cells in 2001 (Elbashir, S. M. et al. (2001) Nature 411, 494-498), it was quickly realized that this sequence-specific mechanism of posttranscriptional gene silencing might be harnessed to develop a new class of medicaments that might also be a promising means for the treatment of diseases not accessible to therapeutic intervention so far (De Fougerolles, A. et al. (2007) Nat. Rev. Drug Discov. 6, 443-453).
However, as RNAi takes place in the cytosol any RNA-based drugs have to pass the cell membrane in order to exert their therapeutic effect. Several methods have been described so far in order to accomplish this goal such as the use of lipids (Schroeder; A. et al. (2010) J. Intern. Med. 267, 9-21), viral carriers (Liu, Y. P: and Berkhout, B. (2009) Curr. Top. Med. Chem. 9, 1130-1143), and polycationic nanoparticles (Howard, K. A. (2009) Adv. Drug Deliv. Rev. 61, 710-720).
Another method for the translocation of molecules through the cell membrane is the use of cell penetrating peptides (CPPs) (also referred to as protein transduction domains (PTDs) or membrane translocation sequences (MTS); reviewed, e.g., in Fonseca, S. B et al. (2009) Adv. Drug Deliv. Rev. 61, 953-964; Heitz, F. et al. (2009) Br. J. Pharmacol. 157, 195-206).
CPPs are a heterogeneous group of peptide molecules—both in terms of their primary amino acid sequences and their structures. Prominent examples of CPPs include the HIV-1 TAT translocation domain (Green; M. and Loewenstein, P. M. (1988) Cell 55, 1179-1188) and the homeodomain of the Antennapedia protein from Drosophila (Joliot; A. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 1864-1868). The exact translocation mechanism is still disputed.
Mutation studies of the Antennapedia protein revealed that a sequence of 16 amino acids called penetratin or pAntp (Derossi, D. et al. (1994) J. Biol. Chem. 269, 10444-10450) is necessary and sufficient for membrane translocation. In the following, other protein-derived CPPs were developed such as the basic sequence of the HIV-1 Tat protein (Vives, E. et al. (1997) J. Biol. Chem. 272, 16010-16017). A synthetic peptide developed is the amphipathic model peptide MAP (Oehlke, J. et al. (1998) Biochim. Biophys. Acta 1414, 127-139).
Coupling of antisense DNA or peptide nucleic acids (PNAs) to CPPs was shown to exert the desired effect in vivo. However, it is still questioned which features were necessary for a CPP to exert its translocation function. In general, little sequence and/or structural resemblance has been found between the different CPPs. So far, the only consistently present feature is the rather high content of basic (positively charged) amino acids resulting in a positive net charge. Thus, it is assumed that CPPs initially bind to negatively charged head groups of lipids or proteins (proteoglycans) in the cell membrane. Once bound, however, the peptides are still inside membrane bound compartments. The further mechanism of uptake is still a matter of extensive debate. It has been proposed that CPPS are either “retrogradely” transported to the ER where they enter the cellular translocation machinery (Fischer, R. et al. (2004) J. Biol. Chem. 279, 12625-12635) or that they directly translocate across the membrane (Rothbard, J. B. et al. (2005) Adv. Drug Deliv. Rev. 57, 495-504). Depending on the mechanism of internalization known CPPs mainly localize in the nucleus or, in case they are internalized in vesicles, mainly remain inside these vesicles, and only a small portion is released into the cytoplasm.
Many CPPs have severe side effects on the cells applied, which is understandable in view of the fact that most of the proteins from which the CPPs are derived function as, e.g., antimicrobial substances or toxins. For example, CPPs can cause cytoplasmic leakage due to membrane disruption and also interfere with the normal functioning of membrane proteins. CPPs might also exhibit cellular toxic effects, such as transportan, which affects GTPase activity (Soomets, U. et al. (2000) Biochim. Biophys. Acta 1467, 165-176). Furthermore, there is a burgeoning body of evidence that many CPPs only exert their function under certain very specific conditions that cannot be met in an in vivo setting. Another drawback is that, depending on the target cell, the CPPs may be rapidly degraded in the cells. Lastly, as many known CPPs are derived from non-human proteins, toxic and/or immunogenic effects have been regularly observed, which may interfere with the utilization of these peptides, e.g., for therapeutic applications in humans.
Thus, there still remains a need for improved cell-penetrating peptides that overcome the above-mentioned limitations. In particular, there is a need for cell-penetrating peptides that represent suitable transfection vesicles or cargos enabling delivery of compounds such as therapeutic agents into target cells with high efficiency but without exerting significant cytotoxic and/or immunogenic effects.
Furthermore, there is also a need for compositions comprising such CPPs as well as for methods employing such CPPs as molecular tools for diagnostic and therapeutic applications.
Accordingly, it is an object of the present invention to provide such CPPs and corresponding compositions and methods.